Secretary of State John Kerry said on Thursday that Jews in a city in eastern Ukraine had been ordered to register with the authorities, calling the idea intolerable.
"Just in the last couple of days, notices were sent to Jews in one city indicating that they had to identify themselves as Jews... or suffer the consequences," Kerry told reporters.
"This is not just intolerable, it's grotesque," he said in Geneva, where he was meeting with his Russian, Ukrainian and European Union counterparts that produced a four-way agreement to work to defuse the crisis in eastern Ukraine.
The origin of the leaflets in Donetsk was unclear and the leader of the pro-Russian separatist group whose letterhead was on the fliers denied any involvement, calling them a "crude forgery" intended to discredit his movement.
Local news site Novosti Donbassa quoted unidentified members of Donetsk's Jewish community saying three masked men handed them out near the city's synagogue on Monday, when Jews were celebrating the start of the Passover holiday.
Purporting to be issued by the Donetsk People's Republic, a group which last week took over public buildings and wants to end rule by the new government in Kiev, the leaflet said all Jews aged over 16 must register with a "commissar" at the regional government headquarters by May 3.
Failure to comply would lead to deportation and the "confiscation of property".
Its preamble explained that action was being taken because Jewish leaders had supported the "junta" which took power in Kiev after the overthrow of the Moscow-backed president.
Kirill Rudenko, a spokesman for the People's Republic of Donbass, said the statement was "complete rubbish": "We made no such demands on Jews," he said. "We have nothing against Jews.
"This is just another attempt to tarnish our image ... It is a crude forgery."
Once home to a large Jewish population that was devastated by the Holocaust, Ukraine has seen a rise in attacks on Jews and on synagogues since unrest began five months ago.
Some Ukrainian nationalist groups which took part in the uprising in Kiev have been blamed for fanning anti-Semitic sentiment. Anti-Semitism is also common among some Russian nationalists.